DNA probe array

ABSTRACT

There is used at least one probe array obtained by arraying particles having various probes, respectively, fixed thereon (probe particles) in a definite order in a holder. A plurality of capillaries or grooves packed with various kinds, respectively, of probe particles are arrayed in parallel, and one of particles contained in each capillary or groove is injected into another capillary or groove to produce a probe array in which the various kinds of probe particles are arrayed in a constant and definite order. Various fluorophore-labeled DNA&#39;s are measured at the same time by attaching various probes to particles, respectively, of different sizes. A probe array composed of various fixed DNA probes can easily be produced, and there can be provided a probe array for detecting various DNA&#39;s which is composed of various fixed arbitrary DNA probes.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a probe array for examiningvarious target at a time when DNA's, RNA's or proteins are substances tobe detected. In particular, it relates to a DNA probe array for DNAdetection by hybridization which has recently been the object ofattention.

[0003] 2. Description of the Related Art

[0004] With the advance of Human Genome Program, there is a strongmovement to diagnose diseases and understand life phenomena byunderstanding living bodies on the basis of DNA. Investigation on theprofile of expressed genes is effective in understanding life phenomenaand investigating the actions of genes. As an effective means forinvestigating the gene expression profile, a DNA probe array obtained byfixing a large number of DNA probes for different kinds of the DNAprobes separately on a solid surface, or a DNA chip has begun to beused. For producing the chip, there is, for example, a process ofsynthesizing an oligomer with a designed sequence base by base in eachof a large number of enclosed cells by employing a photo-chemicalreaction and a lithography widely used in semiconductor industry(Science 251, 767-773 (1991), or a process of spotting DNA probes one byone, respectively, to different cells to make a probe array.

SUMMARY OF THE INVENTION

[0005] The key point of the DNA probe arrays is that they areinexpensive and easy to make any types of probes. The prior art isdisadvantageous in this point. The mass production of the DNA probearrays and the DNA chips requires much labor and time and therefore theyare very expensive. Particularly when the density of the cells where theprobes are fixed, respectively, in a probe array is large, it is gettingdifficult to produce it at a low cost. If the size of each cell for aprobe species is large, such a probe array is easy to produce but isdisadvantageous, for example, in that the volume required for adetection reaction and hence the amounts of samples becomes large as awhole and that measurement with the probe array requires much time andhas no high sensitivity.

[0006] The present invention was made for removing the abovedisadvantages and an object of the present invention is to provide aprocess which permits easy production of a desired DNA probe array(consisting of DNA probes having desired sequences) with a high densityand entails a low production cost.

[0007] A DNA probe array has various DNA probes fixed in separate cells,respectively, and target DNA fragments are detected by hybridizationwith the DNA probes. The target DNA fragments are-labeled with a tagsuch as a fluorophore prior to the hybridization and fluorescence orluminescence and the like are used for the detection of the DNAfragments. The probe array has been used although the density of cellshaving probes therein is not so high. In the conventional probe array,however, the probes are fixed on each cell that spatially divides thesurface of a membrane or the like, the whole area of the probe array isabout 10 cm×5 cm or larger. On the other hand, the size of the newlydeveloped DNA probe array is about 1 cm×1 cm or less although the numberof cells holding DNA probes is very large. The high density probe arrayis constructed on a solid support such as glass or Si wafer which,together with the high density, is good to reduce the amount of samplesconsumed for the hybridization. For example, the size of one cell is assmall as 0.1 mm×0.1 mm, which should be compared to the conventionalsize of 5 mm×5 mm. This high density DNA probe array is called DNA chip.The DNA chip has so many cells holding various probes on the surfaces,respectively. It is used for analyzing multiple components in a sample.In the analysis procedure, at first, all the components in the sampleare labeled with tags such as fluorophores or enzymes. They are placedon the DNA chip for hybridization. If the sample has a component beinghybridized with probes, the component is held on the corresponding cell.By detecting fluorescence the position of the cell emitting fluorescencecan be determined. From the positional information of the fluorescenceemitting cell, the probe species being hybridized with the samplecomponents can be determined. Although the detection and identificationof the hybridized position are easy, the production of DNA chips is notso easy because the probe species required for research or testing arechanging case by case. In addition, the mass production of the chips islabor intensive and expensive. This is mainly due to the high densityproduction cells in a chip. If the density of cells is as low as theconventional one, the production is relatively easy. The presentinventors have found that if the cells can be separately produced andthen assembled to make a probe array, the production becomes easy evenif the probe components should be changed. The change will be carriedout by selecting the cells having probes thereon.

[0008] In order to achieve the above object, in the present invention,solid pieces holding probes, respectively, are composed of smallparticles so as to be movable, and the small particles are sparselyarrayed and then moved to produce a probe array having a densestructure. First, various DNA probes are prepared by synthesis. TheseDNA probes are fixed on the surfaces, respectively, of small particles(beads), so that the kinds of the DNA probes may be different on thedifferent small particles. A large amount of the DNA probes can be fixedon solid surfaces, respectively, by utilizing a method utilizing thecombination of biotin and avidin, a method of fixing DNA probes on Au(gold) surfaces through a SH group (Biophysical Journal 71, 1079-1086(1996), a method of fixing DNA probes on glass surfaces (AnalyticalBiochemistry 247, 96-101 (1997)), a method of fixing DNA probes on anelement matrix of acrylamide gel applied on glass surfaces (Proc Natl.Acad. Sci. USA 93, 4913-4918 (1996)), or the like.

[0009] The various small particles holding the DNA probes on theirsurfaces are placed in a holder for examination in a predetermined orderso as to indicate the kinds, respectively, of the DNA probes, or thesmall particles are arrayed and fixed on a solid surface in apredetermined order so as to indicate the kinds, respectively, of theDNA probes, whereby the probe array is produced. The small particles arespherical such as beads. As to their sizes, their diameters range fromseveral micrometers to 1 mm although depending on purpose of use. Thesmall particles may be square, discoidal or the like, depending onpurpose of use. For usual examinations, spherical beads with a diameterof 0.1 mm to 0.2 mm can be easily used.

[0010] The beads holding the probes, respectively (hereinafter referredto also as “probe beads”) are supplied together with a solvent one byone to a groove for producing probe array. Necessary kinds of probes caneasily be arrayed in the groove, depending on examinations in which theyare used. Since the beads holding the probes, respectively, can beprepared at a low cost, the probe array itself can be produced at a lowcost. These beads having the probes attached thereto which have beenarrayed in the groove are used after being placed in a capillary forexamination or a cell having a narrow space. The employment of acapillary as a probe array holder is advantageous in that the amounts ofsample DNA's to be examined can be reduced. It is advantageous also inthat the capillary can easily be connected to a solvent-introducingsystem.

[0011] On the other hand, when solid particles having the probes,respectively, fixed thereon are made distinguishable from one another,there is such an advantage that the trouble of arraying the probes by adefinite method can be saved.

[0012] As explained above, according to the present invention, anarbitrary probe array can be produced easily at a low cost. Moreover, aprobe array which reduces the amount of reagents and permits easyinjection of the reagents and easy washing can be provided by itsconstruction in a capillary.

[0013] Typical examples of the present invention are summarized below.In the typical examples of the present invention, there is used at leastone probe array obtained by arraying particles having various probes,respectively, fixed thereon (hereinafter referred to also as “probeparticles”) in a definite order in a holder. A plurality of capillariesor grooves packed with various kinds, respectively, of probe particlesare arrayed in parallel, and one of particles contained in eachcapillary or groove is injected into another capillary or groove toproduce a probe array in which the various kinds of probe particles arearrayed in a constant and definite order various fluorophore-labeledDNA'S are measured at the same time by attaching various probes toparticles, respectively, of different sizes. The present inventionpermits easy production of a probe array composed of various fixed DNAprobes, and provides a probe array for detecting various DNA's which iscomposed of various fixed arbitrary DNA probes.

[0014] There are summarized below characteristics of the DNA probe arrayfor examining many items of the present invention and a process forproduction thereof.

[0015] (1) A probe array for examining many items which comprises anarray of a plurality of particles having probes, respectively, fixedthereon, said probes being capable of binding to different targetsubstances to be examined (e.g. DNA's, proteins or the like),respectively.

[0016] (2) A probe array for examining many items which comprises aplurality of particles having probes, respectively, fixed thereon, saidprobes being capable of binding to different target substances to beexamined, respectively, wherein said particles are arrayed in a line ina predetermined order, and said order is such that the arrayingpositions of said particles correspond to the kinds, respectively, ofsaid probes fixed on said particles.

[0017] (3) A probe array according to the item (2), wherein the sizes orshapes of said particles holding said probes correspond to the kinds,respectively, of said probes fixed on the surfaces of said particles.

[0018] (4) A probe array according to the item (2), wherein saidparticles holding said probes are labeled with different dyes orfluorophores, respectively, depending on the kinds of said probes heldby the particles.

[0019] (5) A probe array according to the item (2), wherein said probesare arrayed to form a layer on a two-dimensional plane.

[0020] (6) A probe array according to the item (2), wherein saidparticles are one-dimensionally arrayed, and the order of particles (,therefore the probes), is predetermined.

[0021] (7) A probe array according to the item (2), wherein saidparticles are held in a container having a transparent window.

[0022] (8) A probe array according to the item (2), wherein saidparticles holding said probes are held in a capillary.

[0023] (9) A probe array according to the item (2), wherein saidparticles holding said probes are held in a groove formed on a flatsolid surface or a groove formed between two flat surfaces.

[0024] (10) A probe array according to the item (2), wherein saidparticles holding said probes are two-dimensionally arrayed at apredetermined position(s) by arraying a plurality of capillaries holdingsaid particles holding said probes, or by arraying said particlesholding said probes in grooves formed on a flat surface.

[0025] (11) A probe array according to the item (2), wherein saidparticles holding said probes are held in a gel-like substance.

[0026] (12) A probe array for examining many items which comprises aplurality of particles having probes, respectively, fixed thereon, saidprobes being capable of binding to different target substances to beexamined, respectively, wherein said particles are arrayed so thatcharacteristics of said particles may correspond to the kinds,respectively, of said probes.

[0027] (13) A probe array according to the item (12), wherein the sizesor shapes of said particles holding said probes correspond to the kinds,respectively, of said probes fixed on the surfaces of said particles.

[0028] (14) A probe array according to the item (12), wherein saidparticles holding said probes are labeled with different dyes orfluorophores, respectively, depending on the kinds of said probes heldby the particles.

[0029] (15) A probe array according to the item (12), wherein saidprobes are arrayed to form a layer on a two-dimensional plane.

[0030] (16) A probe array according to the item (12), wherein saidparticles are one-dimensionally arrayed.

[0031] (17) A probe array according to the item (12), wherein saidparticles are held in a container having a transparent window.

[0032] (18) A probe array according to the item (12), wherein saidparticles holding said probes are held in a capillary.

[0033] (19) A probe array according to the item (12), wherein saidparticles holding said probes are held in a groove formed on a flatsolid surface or a groove formed between two flat surfaces.

[0034] (20) A probe array according to the item (12), wherein saidparticles holding said probes are two-dimensionally arrayed at apredetermined position(s) by arraying a plurality of capillaries holdingsaid particles holding said probes, or by arraying said particlesholding said probes in grooves formed on a flat surface.

[0035] (21) A probe array according to the item (12), wherein saidparticles holding said probes are held in a gel-like substance.

[0036] (22) A process for producing a probe array which comprises a stepof fixing probes on the surfaces, respectively, of particles, and a stepof arraying a plurality of said particles having said probes fixedthereon.

[0037] (23) A process for producing a probe array according to the item(22), wherein said particles are arrayed on a straight line in apredetermined order so as to indicate the kinds, respectively, of saidprobes fixed on said particles.

[0038] (24) A process for producing a probe array according to the item(22), wherein the plurality of said particles having said differentprobes, respectively, fixed thereon are transferred to a groove or probearray holder for arraying said particles, by using a plurality ofcapillaries or grooves for transferring said particles having saidprobes fixed thereon, and said particles are arrayed on a straight linein a predetermined order so as to indicate the kinds, respectively, ofsaid probes fixed on said particles.

[0039] (25). A process for producing a probe array which comprises astep of fixing probes on the surfaces, respectively, of particles, and astep of arraying a plurality of said particles having said probes fixedthereon, as a mixture on a plane, wherein the kinds of said probes fixedon said particles are distinguished by the shapes or sizes or any otherphysical or chemical properties of the particles or fluorophoreslabeling said particles, respectively.

[0040] (26) A process for producing a probe array according to the item(25), wherein the plurality of said particles having said differentprobes, respectively, fixed thereon are transferred at the same time toa groove or probe array holder for arraying said particles, by using aplurality of capillaries or grooves for transferring said particleshaving said probes fixed thereon.

[0041] (27) A process for producing a probe array according to the item(25), wherein said particles having said probes fixed thereon are heldin different particles reservoirs for the different kinds of saidprobes, supplied one from each reservoir to a groove for arraying saidparticles, through a capillary or a groove to be arrayed, andtransferred to a probe array holder while maintaining the array, wherebya probe array is produced.

[0042] (28) A process for producing a probe array according to the item(25), wherein said particles having said probes fixed thereon are heldin different particles reservoirs for the different kinds of saidprobes, supplied one at a time from each reservoir to a groove forarraying said particles, through a capillary or a groove to be arrayed,and transferred to a probe array holder by means of an electric forcewhile maintaining the array, whereby a probe array is produced.

[0043] (29) A process for producing a probe array according to the item(25), wherein said particles having said probes fixed thereon are heldin different particles reservoirs for the different kinds of saidprobes, supplied one at a time from each reservoir to a groove forarraying said particles, through a capillary or a groove to be arrayed,and transferred to a probe array holder by means of a solution flowwhile maintaining the array, whereby a probe array is produced.

[0044] (30) A method for detecting target substances to be examinedwhich bind to probes, respectively, held on the surfaces, respectively,of particles, said method comprising a step of labeling said targetsubstances with a fluorophore or a material emitting phosphorescence orany tag and a step of irradiating said particles with light (laserbeams), followed by optical detection of the fluorescence orphosphorescence emitted.

[0045] (31) A method for detecting target substances to be examinedaccording to the item (30), wherein a light source (laser beams) isscanned along a straight line on which said particles are arrayed, andsaid fluorescence or phosphorescence emitted from tags is detected withan optical sensor.

[0046] (32) A method for detecting target substances to be examinedaccording to the item (30), wherein said light (laser beams) is castedalong a straight line on which said particles are arrayed, and saidfluorescence or phosphorescence emitted from each of the positions atwhich said particles, respectively, are arrayed, is detected.

[0047] (33) A method for detecting target substances to be examinedaccording to the item (30), wherein a pattern of scattering of saidlight (laser beams) by said particles is obtained by the irradiationwith said light (laser beams), fluorescence or phosphorescence emittedfrom said target substances binding to said probes fixed on saidparticles is detected, the shapes of said particles or fluorescencesemitted by the fluorophores labeling said particles, respectively, aredetected, whereby the amounts of said target substances attached to saidprobes are determined.

[0048] (34) A method for detecting target substances to be examinedaccording to the item (30), wherein said particles are detected whilebeing allowed to flow.

[0049] (35) A method for detecting target substances to be examinedaccording to the item (30), wherein said target substances attached tosaid probes fixed on said particles are measured as fluoroscopic images.

[0050] (36) A method for detecting target substances to be examinedaccording to the item (30), wherein said particles are measured astwo-dimensional images.

[0051] (37) A process for producing a probe array which comprises afirst step of fixing probes on the surfaces, respectively, of particles,a second step of dividing a plurality of said particles having saidprobes fixed thereon, into groups and arraying particles in each groupin a compartment on a solid surface, and a third step of reacting saidprobes fixed on said particles with target substances to be examined, insaid compartments, wherein the state of distribution of said particlesin the first step is different from the state of distribution of saidparticles in said compartments where said third step is carried out.

[0052] (38) A probe array which comprises an array of a plurality ofsmall particles having probes, respectively, thereon, wherein saidprobes are arrayed in one-dimensionally or two-dimensionally, andwherein an order of arrangement of small particles having probes ispredetermined, or positions of arrangement of small particles havingprobes are predetermined.

[0053] (39) A probe array according to the item (38), wherein markerparticles are placed between the small particles having different kindsof probes. The marker particles are labeled with fluorophores differentfrom the fluorophores labeling the small particles, and the positions ofthe marker particles on the probe array are reference positions fordiscriminating the species of the probes on the small particles.

[0054] (40) A probe array according to the item (38), wherein species ofthe probes on each of the small particles are different from each other.

[0055] (41) A probe array according to the item (38), wherein the smallparticles include particles having same species of the probes.

[0056] (42) A probe array according to the item (38), wherein each ofthe probes is capable of binding to DNA, RNA or a protein.

[0057] (43) A probe array according to the item (38), wherein the smallparticles are spherical beads with an outer diameter of 1 μm to 10 μm.

[0058] (44) A probe array according to the item (38), wherein the smallparticles are spherical beads with an outer diameter of 10 μm to 100 μm.

[0059] (45) A probe array according to the item (38), wherein the shapeof the small particles is a cubic shape.

[0060] (46) A probe array according to the item (38), wherein the shapeof the small particles is a cylindrical shape.

[0061] (47) A probe array according to the item (38), wherein the smallparticles are made of glass or plastics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0062]FIG. 1 is a diagram showing a production process of a DNA probearray of a first example of the present invention and an examinationapparatus using the DNA probe array.

[0063]FIG. 2A is a plane view of a plate having fine grooves which is apart of a jig for producing a probe array using small particles as aprobe-fixing medium, in the first example of the present invention, andFIG. 2B is a cross-sectional view of the jig for producing the probearray.

[0064]FIG. 3 is a diagram showing an example of probe array holder inthe first example of the present invention.

[0065]FIG. 4 is a schematic diagram showing an example of an apparatususing a DNA probe array, in which one of the linear probe array and alight source is scanned in relation to the other, in the first exampleof the present invention.

[0066]FIG. 5 is a schematic diagram showing an example of an apparatususing a DNA probe array, in which laser beams are casted along a lineararray of fine particles, in the first example of the present invention.

[0067]FIG. 6 is a schematic diagram showing an example of an apparatususing a DNA probe array, in which light is casted on the whole of aregion where the probe array is present, in the first example of thepresent invention.

[0068]FIG. 7 is a diagram showing an example of results which areobtained by means of the structure shown in FIG. 6 using a smallparticle type probe array, and are outputted in a monitor, in the firstexample of the present invention.

[0069]FIG. 8A is a plane view of a jig for introducing small particleholding probes, respectively, into a two-dimensional probe array holder(a holder having probes arrayed in two-dimensional area) 34, in a secondexample of the present invention, and FIG. 8B is a cross-sectional viewof the jig.

[0070]FIG. 9 is a schematic illustration of an example of method forintroducing small particles holding probes, respectively, into a groovefor arrayment from small-particles reservoirs, in a third example of thepresent invention.

[0071]FIG. 10 is a schematic diagram showing the structure of anexamination apparatus in which samples are detected by detection of manycolors by the use of a two-dimensional probe array, in a fourth exampleof the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The examples of the present invention are explained below indetail with reference to the drawings.

FIRST EXAMPLE

[0073]FIG. 1 is a diagram showing a production process of a DNA probearray of the first example of the present invention and an examinationapparatus using the DNA probe array. Spherical plastic particles (beads)1 (diameter: 0.2 mm) holding avidin 70 on their surfaces are prepared.The precision of diameter of the small particles 1 is 5%. A DNA probeobtained by PCR amplification by using biotin-attached primers isseparated into individual strands, and the resulting biotin 71-attachedDNA probe 2 is combined with the small particle holding avidin. Thus,each kind of DNA probes are captured by the small particles,respectively, whereby a plurality of groups of small particles attachedwith probe 3 are formed. Needless to say, synthetic DNA strands may beused as DNA probes. In this case, the DNA probes can be directlyattached to solid (small particle) surfaces, respectively, without thecombination of biotin and avidin. Methods for attaching DNA probes tosolid surfaces, respectively, are described, for example, in theabove-mentioned references (Biophysical Journal 71, 1079-1086 (1996) andAnalytical Biochemistry 247, 96-101 (1997)). The following method mayalso be employed: a specific sequence of nucleotides all of which arethe same, for example, TTTTT . . . TT is fixed on each solid (smallparticle) surface, and each DNA oligomer having a poly A strand ishybridized with the nucleotide sequence to be bonded thereto by bindingbetween complementary strands, whereby the DNA oligomer is introducedonto the solid (small particle) surface.

[0074] The thus prepared small particles holding the probes,respectively, are arrayed one by one in a transparent capillary tube (aprobe array holder 7) to obtain a probe array 4. The kind of the probeheld on the surface of each small particle can be known from the placeof this small particle in the order of the small particles arrayed inthe capillary tube (the probe array holder 7). Therefore, afterhybridization between the DNA probes 2 and sample DNA's having afluorophore tag 8 attached thereto (numeral 9 shows a sample DNAfragment captured by the small particle 1 by binding betweencomplementary strands), followed by irradiation with light, the kinds ofDNA's in a specimen can be known from the fluorescence emitted.

[0075] Sample DNA's having a fluorophore tag 8 attached thereto isinjected into the capillary tube (the probe array holder 7) containingthe probe array 4 composed of probes 1, 2, . . . , n, through the sampleinlet 5 of the tube in the sample inflow direction 6 to hybridize theDNA probes 2 with the sample DNA's having the fluorophore tag 8 attachedthereto. Then, the probe array holder 7 is set on the movable table (notshown) of the apparatus and laser beams from a laser source 11 arefocussed by a lens 12 and casted on the moving probe array holder 7.Numeral 5″ shows a sample outlet. Fluorescence emitted from sample DNA'shaving the fluorophore tag 8 attached thereto which have been capturedby the small particles 1 at positions irradiated with the laser beams iscolor (a wavelength) selectively detected by a filter 12 and thephotodetector of a CCD (charge coupled device) camera 13 which detectsthe fluorescence from a direction substantially perpendicular to thedirection of the laser beams irradiation. The fluorescence signals thusdetected are displayed in real time on a monitor 17. By a dataprocessing unit 15, they are processed to obtain fluorescence intensityemitted from particles. The results are displayed on a display unit 16.The axis of abscissa of an output pattern displayed in the monitor 17refers to the positions of the small particles 1 arrayed in thecapillary tube (the probe array holder 7) and hence the kinds of theprobes, and the axis of ordinate refers to fluorescence intensity whichindicates the presence of the sample DNA fragment bonded to any of theprobes by binding between complementary strands. A controller 14controls the movement of the above-mentioned movable table, theincorporation of signals from the CCD camera 13 and the transmission ofsignals to the data processing unit 15 and the monitor 17. Whether anobjective base sequence is present in any of the sample DNA's (DNAfragments) or not can be judged from the output in the monitor 17 or thedisplay unit 16.

[0076] It is also possible to carry out the detection by allowing beadsto flow together with a solvent instead of moving the capillary tubeholding beads.

[0077] Although the fluorophore tag is attached to the sample DNA's (DNAfragments) in the above explanation, it is also possible to attachdifferent fluorophore tags to the probes 1, 2, . . . , n, respectively,instead of attaching the fluorophore tag to the sample DNA's (DNAfragments). In this case, the filter 12 is composed of a many-colorfilter capable of selecting a plurality of wavelength regions, orwavelengths of fluorescence are separated by using optical prism, adiffraction grating or the like.

[0078] Next, a process for producing a probe array using small particlesas a probe-supporting medium, in First Example is explained below.

[0079]FIG. 2A is a plane view of a plate having fine grooves which is apart of a jig for producing a probe array using small particles as aprobe-supporting medium, and FIG. 2B is a cross-sectional view of thejig for producing the probe array. Small particles 1 having probes,respectively, attached thereto can be arrayed by means of a device forarraying fine particles by the use of fine grooves. The plate 18 havingfine grooves for arraying small particles which is shown in the planview in FIG. 2A is used after being equipped with the transparent cover23 which is shown in the cross-sectional view in FIG. 2B. The plate 18has the following grooves formed thereon: a plurality of grooves 19 forarraying small particles holding different kinds, respectively, ofprobes in different grooves, respectively; a groove for arraying variousprobe-holding small particles (a fine groove for producing probe array)20 which intersects (is perpendicular to) the grooves 19; and groovesfor outlet of solution 21 which discharge a solution. The maximum widthsand maximum depths of the grooves 19 and the groove 20 are adjusted tovalues at which two small particles cannot enter the same groove(namely, the maximum widths and maximum depths satisfy the condition 1that they should be less than 2R when the diameter of the small particleis taken as R). The maximum width and maximum depth of the grooves 21are adjusted to values at which the small particles cannot pass thegrooves 21 (namely, the maximum width and maximum depth satisfy thecondition 2 that they should be less than R when the diameter of thesmall particle is taken as R). That is, the small particles 1 can passthrough capillaries formed by the transparent cover 23 and the finegrooves 19 and 20 formed on the plate 18. As the forms of section of thegrooves 19, 20 and 21, any forms may be employed so long as they satisfythe above conditions 1 and 2.

[0080] In each of the fine grooves 19, small particles holding the samekind of probes, respectively, are arrayed at random distance intervals.Thus, small particles holding different kinds of probes are divided intogroups which are held in the fine grooves 19, respectively. For example,small particles each holding a probe 1 are arrayed in the first groove19-1 among the grooves 19, small particles each holding a probe 2 in thesecond groove 19-2, . . . , small particles each holding a probe n inthe n-th groove 19-n. Various probe-holding small particles are arrayedin the groove 20 perpendicular to the plurality of the grooves 19. Thesmall particles 1 having probes, respectively, attached thereto areintroduced into the groove for making an array 20 by a solution flow oran electric field. Since two small particles cannot enter the groove 19sideways because of their size, each of small particles holding variousprobes, respectively (probe particles) is arrayed at an intersection ofthe array of plurality of the fine grooves 19 and the groove forarraying probes 20. The grooves 21 after the intersection are so thinthat the particles cannot go forward. At this point of time, thedistances between two particles are random. The particles arrayed in thegroove 20 are introduced into a probe array holding capillary (a probearray holder 7) (inside diameter: 0.3 mm) by a solution flow or anelectric field in a direction perpendicular to the grooves 19, namely,along the groove 20 for making an array, to be closely arrayed. Numeral22 shows a probe array holder connector (a guide device for connectingthe probe array and solution inlet as well as outlet) which connects adevice for arraying small particles (23 and 20) and the probe arrayholder 7. Numeral 5′ shows a stopper tube. Particles having DNA probes,respectively, fixed thereon which are to be used are supplied toparticles reservoirs (see 38 in FIG. 9) communicating with the grooves19, respectively. The arraying order of the particles reservoirs holdingthe particles having the probes fixed thereon corresponds to thearraying order of the probes in the groove 20 and the probe arrayholding capillary. It is also possible to insert a marker between theparticles having the probes fixed thereon, for making it easy to knowthe order.

[0081] Next, there is explained an example of the probe array holder 7which holds probes in a capillary tube, in First Example.

[0082]FIG. 3 is a diagram showing an example of probe array holder inFirst Example. Small particles having probes, respectively, attachedthereto are held in a probe array holder 7 (a capillary) having a sampleinlet and a sample outlet. A terminal adoptor (a terminal adoptor forcapillary holder and the solution outlet) 24 is attached to each end ofthe probe array holder 7 through a stopper tube 5′ and a probe arrayholder connector (a guide device for connecting the probe array andsolution inlet as well as outlet) 22 in order to prevent the outflow ofthe small particles 1. Needless to say, the adoptor 24 is attached afterintroducing the small particles into the capillary (the probe arrayholder 7).

[0083] DNA samples to be examined are labeled with a fluorophore (inthis case, Cy-5 (maximum emission wave-length: 650 nm) is used) andintroduced together with a solvent into the capillary holding the probearray (the probe array holder 7) to cause hybridization between the DNAsamples and the probes. After target DNA's are captured on some of theprobes respectively, by the hybridization, the excess DNA samples arewashed away, followed by detection of fluorescence. The linear probearray is advantageous in that the probe array holder 7 holding the probearray is easy to scan mechanically, resulting in low consumption of thesamples. The fluorophore tag includes Texas Red (maximum emissionwavelength: 615 nm), fluorescein isocyanate (maximum emissionwavelength: 520 nm), etc. In addition to these fluorophore tags, tagscapable of emitting phosphorescence may be used. After the unreacted DNAis washed away, the residue is introduced into a measuring apparatus.The measuring apparatus is composed of a laser for excitation and afluorescence detector. A large number of the small particles areirradiated at the same time by scanning laser beams along the capillarytube (the probe array holder) or casting laser beams on the interior ofthe capillary tube along the tube, and the resulting fluorescence imagesare detected. It is also possible to introduce the small particles oneafter another into an irradiation portion by moving the capillary tube.In addition, the measurement may be carried out while jetting thesolvent and the beads (small particles) through a nozzle, as in a cellsorter.

[0084] Next, an example of an apparatus using a DNA probe array isexplained below.

[0085]FIG. 4 is a schematic diagram showing an example of an apparatususing a DNA probe array, in which one of the linear probe array and alight source are scanned in relation to the other. In FIG. 4, the samestructure as shown in FIG. 1 is employed, and laser irradiation iscarried out as follows: a laser irradiation position and a detector 13are fixed and a probe array holder 7 holding probes is moved in relationto them; or the probe array holder 7 is fixed, and the laser irradiationposition and the detector 13 are moved in relation to the probe arrayholder 7. As the detector 13, a photomultiplier or a lensequipped cooledCCD camera is used. Fluorescence is detected from a directionsubstantially perpendicular to the laser irradiation direction.

[0086]FIG. 5 is a schematic diagram showing an example of examinationapparatus using a DNA probe array, in which laser beams are casted alonga linear array of fine particles. In FIG. 5, laser beams from a lasersource 11 are casted along the axis of a probe array holder 7 in thedirection of said axis. Fluorescence emitted by a fluorophore tag iscondensed in a microlens-array (Cell Fock Lens (a trade name of NipponSheet Glass Co., Ltd.)) 25 and projected on a line sensor (a CCD linesensor) 27 through a filter 26. The other constituents shown in FIG. 5are the same as those in the structure shown in FIG. 1. Although thestructure shown in FIG. 5 is effective, it can be employed only when thesmall particles are transparent.

[0087]FIG. 6 is a schematic diagram showing an example of examinationapparatus using a DNA probe array, in which light is casted on the wholeof a region where the probe array is present (a region where smallparticles having probes are held and irradiated with laser) 30. Althoughthe probe array is one-dimensional in the structure shown in FIG. 6, acooled CCD area sensor or the like is used for fluorescence detection sothat the examination apparatus can be used also when the probe array istwo-dimensional. Laser beams from a laser source 11 change their courseat a mirror 28 and the region of irradiation with the laser beams isone-dimensionally expanded by a beam expander 29, whereby the laser beamis casted on the whole of the region 30 where small particles havingprobes are held and irradiated with laser, in a probe array holder 7.When a two-dimensional photodetector such as a CCD area sensor is used,two-dimensional fluorescence images are obtained and the kinds of probesare known from the emission positions. The other constituents shown inFIG. 6 are basically the same as in FIG. 1. In FIG. 6, a confocalmicroscope or a similar technique may be used. For example, instead ofusing the beam expander 29, laser scanning is carried out by changingthe course of the laser beams by rotating the mirror 28, andfluorescence emitted by a fluorophore tag present at a position in theprobe array holder on which the laser beams are casted.

[0088]FIG. 7 is a diagram showing an example of results which areobtained by means of the structure shown in FIG. 6 using a smallparticle type probe array, and are output in a monitor 17. In theexample shown in FIG. 7, a small particle emitting fluorescence (a smallparticle attached with target DNA) 31 is shown by a closed circle, and asmall particle not emitting fluorescence (a small particle not attachedwith target DNA) 32 is shown by an open circle. The fluorescenceintensity is high at the closed circles 31, indicating that DNA's arecaptured by probes. Although sample DNA's are labeled with a singlefluorophore in the example shown in FIGS. 6 and 7, it is also possibleto label a plurality of specimens for examination of DNA's with aplurality of fluorophores, respectively, and carry out comparativemeasurements.

[0089] As explained above, when DNA probes held by particles are held ina capillary, there are advantages such as easy supply of samples andeasy washing. Moreover, there are advantages such as easy fluorescencemeasurement, easy production of a necessary desirable probe array, andprovision of an inexpensive probe array. In addition, the volume ofsamples which is required for hybridization can be reduced. Although acapillary is used for holding a plurality of probes in First Example, agroove formed on a transparent and flat plate may also be used. Whenparticles are arrayed in the groove, the following is possible: a gel isheld in the bottom of the groove, and particles holding probes,respectively, are arrayed in the groove and then fixed on the gel bypressing thereon. In this case, a space to be filled with a samplesolution is reduced, so that the consumption of the samples can bereduced. Needless to say, also when a probe array is produced using atransparent and flat plate having a groove formed thereon, the exampleof examination apparatus shown in any of FIG. 1, FIG. 4, FIG. 5, FIG. 6and FIG. 10 (hereinafter described) can be used, and samples captured byprobes can easily be detected by fluorescence.

SECOND EXAMPLE

[0090] In First Example, the probe array is formed on a straight line.In Second Example, an example of two-dimensional arrangement of probearrays is given. Small particles used in Second Example have the samediameter of 0.2 mm as that in First Example. When particles with adiameter of 0.1 mm or 0.05 mm or less than 0.05 mm are used, it isnecessary to change the pitch and depth of grooves and the size of aprobe array holder which are described hereinafter. Particles of varioussizes ranging from 1 μm to 100 μm in diameter are supplied for the use.

[0091] For the two-dimensional arrangement of probe arrays, there isused either a probe array holder obtained by arraying capillariesholding probe arrays, respectively, or a probe array holder obtained byforming a plurality of grooves on a flat plate and attaching atransparent cover to the flat plate. A process for producing a probearray holder for a two-dimensional array of probe arrays by arrayingcapillary tubes is the same as the production process of a probe arrayholder in First Example, except that a plurality of capillaries aremerely arrayed. However, a housing for holding the plurality of thecapillaries in parallel, supplying samples and introducing a wash liquidshould be modified so as to be suitable for the multi-capillary. Whenlaser beams are casted on the plurality of the capillaries, it ispreferable to adopt either a method comprising casting laser beams froma direction parallel to a plane on which the capillaries are arrayed,and detecting fluorescence emitted in each capillary with atwo-dimensional detector, or a method comprising casting laser beamstwo-dimensionally expanded by a beam expander, with scanning, orcarrying out light irradiation by light scanning of laser beamscondensed into a point, and detecting fluorescence emitted in eachcapillary with a two-dimensional detector.

[0092] A probe array holder obtained by forming a plurality of grooveson a flat plate is explained below. A two-dimensional probe array holder(a holder having probes arrayed in two-dimensional area) 34 is composedof a cell having a space of 0.1 mm between two transparent flat platesthe lower of which has grooves. Each groove has a pitch of 0.3 mm, awidth of 0.25 mm and a depth of 0.15 mm. Both the width and depth of thegroove are too small for the passage of two particles and are sufficientfor the passage of one particle. Small particles having probes,respectively, fixed thereon are arrayed in each groove. The smallparticles have a diameter of 0.2 mm and cannot move from one groove toanother groove.

[0093]FIG. 8A is a plane view of a jig 33 for producing atwo-dimensional probe array for introducing small particle holdingprobes, respectively, into a two-dimensional probe array holder (aholder having probes arrayed in two dimensional area) 34, and FIG. 8B isa cross-sectional view of the jig. For arraying small particles in thetwo-dimensional probe array holder 34, guide grooves 36, guide groovesfor arraying small particles in cell are used. The openings of thegrooves 36 are covered with a transparent cover 35. As shown in FIG. 8A,each groove 36 is widen toward the end, and small particles (beads) 1having probes, respectively, attached thereto are arrayed in the wideportion of the groove 36 in a predetermined order so as to indicate thekinds, respectively, of the probes held by the small particles. Thesmall particles. 1 having probes attached thereto which have beenarrayed in the array of the grooves 36 are moved to the narrow portions(particle-holding portions) of the grooves 36 by a solvent flow or anelectric field to be transferred to the two-dimensional probe arrayholder 34. As shown in the cross-sectional view shown in FIG. 8B, thesmall particles having probes attached thereto which have been sparselyarrayed in the grooves 36 are held in the two-dimensional probe arrayholder 34 in a closely arrayed state. The probe array holder 34 hasopenings (not shown) for introducing and draining, respectively, asample solution or a wash liquid. The above arrayment of the smallparticles having probes attached thereto in the grooves 36 is carriedout by, as in First Example, arraying the small particles in capillariesat first and transferring them to the grooves 36. Since small particlesin any of the grooves 36 cannot move to the adjacent groove 36,different groups of probe particles are arranged in different arrays(different grooves). The order of the probes in each array (each groove)can be determined in the same manner as in First Example. Then, thesmall particles are transferred to fine grooves 37, fine grooves in cellfor keeping fine particles attached with probe, from the grooves 36.

[0094] The apparatus shown in FIG. 6 is modified for detectingtwo-dimensional probe array holder 34. In the modified apparatus, asshown in FIG. 6, one-dimensionally expanded laser beams are linearlycasted on the two-dimensional probe array holder 34, and laser beams arelinearly scanned in a direction perpendicular to the direction of theexpansion. The two-dimensional fluorescence images thus obtained aredetected using a two-dimensional detector (e.g. CCD). Needless to say,it is also possible to obtain two-dimensional fluorescence images bytwo-dimensionally scanning laser beams condensed into a point.

THIRD EXAMPLE

[0095] A key point of the process of the present invention is a methodfor easily arraying small particles. In Third Example, there isdescribed an example of production of a probe array by easy method toarray small particles. In a probe array, close array of solid pieces(particles) holding probes, respectively, is important in reducing thereaction volume and facilitating the measurement. On the other hand, theproduction of a probe array is easier when the probe-holding positionsare scattered. Accordingly, the point of the present invention is toemploy a structure in which the probe-fixing positions are movable, anddensify the structure after the formation of a probe array. That is, thepoint of the present invention is to move solid pieces (particles)having probes, respectively, fixed thereon, and thereby produce a probearray composed of a tight array of the solid pieces (particles) havingprobes fixed thereon.

[0096] For arraying small particles holding various probes,respectively, on their solid surfaces (probe particles), there is amethod of arraying the small particles one by one in the groove of aprobe array holder with tweezers or the like. This method is similar toa method employed for producing a probe array by attaching probes to theseparate cells, respectively, on a continuous solid surface. Thisso-called spotting becomes more difficult as the region of each probebecomes finer. However, it is possible to finely array independent smallparticles having probes, respectively, attached thereto. For example, toproduce a probe array composed of a tight array of solid pieces(particles) having probes, respectively, fixed thereon, the following issufficient: first, small particles having probes, respectively, attachedthereto are sparsely placed in the grooves shown in FIG. 2 or thegrooves shown in FIG. 8, and are moved by a solution flow, an electricfield or the like to form a tight array, which is held in a probe arrayholder 7 or 34.

[0097]FIG. 9 is a schematic illustration of an example of method forintroducing small particles holding probes, respectively, into a groovefor arrayment from small-particles reservoirs. In the probe arrayproduction process shown in FIG. 9, from each reservoir holding a largenumber of the same kind of small particles having probes, respectively,attached thereto, the small particle is supplied to a groove forarrayment. In FIG. 9, the small particle 1 is supplied from eachreservoir 38 of the small particles 1 to a groove for arrayment (20 inFIG. 2) through fine grooves (19 in FIG. 2) by a solution flow to betransferred to a probe array holder 7. The following is also possible:similarly, the small particle 1 is supplied from each reservoir 38 ofthe small particles 1 to a groove for arrayment (36 in FIG. 8) by asolution flow to be transferred to a probe array holder 34. Thereservoirs 38 of the small particles may be perpendicular to or on aplane on which the grooves 19 or 36 are formed, though in Third Example,a convenient structure can be obtained when the particles reservoirs 38are perpendicular to the plane and are mountable and demountable. Thisis because the kinds of the probes can be freely changed depending onpurposes. In the particle array, the probe species on the particles arerecognized by their positions in the array.

FOURTH EXAMPLE

[0098] In Fourth Example, small particles having probes, respectively,attached thereto are arrayed according to the predetermined order andthe probe species can be recognized by the particle positions. In Firstto Third Examples, the kinds of probes held by small particles,respectively, are distinguished by the positions at which the smallparticles are held, respectively, though Fourth Example discloses amethod for distinguishing the kinds not by the positions of smallparticles under observation but by the shapes (e.g. particle diameter),dielectric properties, electromagnetic properties or colors of the smallparticles themselves. That is, this method is such that in obtainingsignals from fluorophore-labeled samples captured by probes, the kindsof the probes on the surfaces of particles are investigated at the sametime by measuring the shapes, colors or the like of the particles. It isalso possible to label particles with one or more fluorophores differentin kind from the fluorophore labeling the samples.

[0099] In Fourth Example, there is described a case of labeling sampleswith a long-life fluorophre or phosphor tag, and distinguishing smallparticles by the differences among their sizes and the difference amongfluorescences emitted by fluorophores labeling the small particles,respectively. As the fluorophores for distinguishing the smallparticles, there are used fluorophores having a relatively shortfluorescence life, such as FITC, Texas Red, Cy-5, etc. The reason whythe particles are labeled with the fluorophores having a relativelyshort fluorescence life is that the distinction of fluorescences emittedby the fluorophores labeling the particles from fluorescence emitted bythe fluorophore with a long fluorescence life used for labeling thesamples is made possible. Needless to say, it is also possible to carryout the distinction by changing the fluorescence wavelengths.

[0100]FIG. 10 is a schematic diagram showing the structure of anapparatus in which samples are detected by detection of many colors bythe use of a two-dimensional probe array. FIG. 10 shows a case of usinga plurality of filters. First, light from a flash lamp 40 is casted on aprobe array 39 composed of a two-dimensional array of small particles 1having probes, respectively, attached thereto which have been reactedwith samples, the light transmitted by a transparent supporting tablefor the probe array 39 is passed through a light attenuation filter ifnecessary, signals detected by a CCD (charge coupled device) camera 13are input to a data processing unit 15, it is confirmed that theparticles do not overlap in the probe array 39, and the shapes of theparticles (beads) are measured.

[0101] Subsequently, laser beams 11 from a laser source are casted onthe probe array 39 by a rotary mirror 28 capable of scanning the laserbeams in a first direction and a beam expander 29 capable of expandingthe irradiation region of the laser beams in a second directionperpendicular to the first direction. Fluorescences having variouswavelengths are selected and separated while changing the transmissionwavelength by sliding a plurality of fluorescent wavelength selectivefilters 42 held by a slide-type or rotary-type filter holder 41. Afterbeing separated, the fluorescences emitted from the probe array 39 aredetected by the CCD camera 13 or array detector. The controller 14controls the rotary mirror, the flash lamp 40, signal incorporation fromthe CCD camera 13, and signal transmission to the data processing unit15 and a monitor 17. Whether an objective base sequence is present inany of the sample DNA's (DNA fragments) can be judged from the output inthe monitor 17 or a display unit 16.

[0102] In Fourth Example, mixtures of two fluorophores in various ratiosare attached fast to surfaces, respectively, of small particles. Thus,20 or less small particles are distinguished by mixtures of each pair offluorophore by varying the mixing ratio. For example, when fluorophoresF1 and F2 are used and the mixing ratio between these fluorophores F1and F2 is taken as (w1, w2), 20 ratios are selected as the mixing ratio(w1, w2) from the following ratios: (w1, w2) =(0, 0.05), (0.05, 0.95),(0.1, 0.9), (0.15, 0.85), (0.2, 0.8), (0.25, 0.75), (0.3, 0.7), (0.35,0.65), (0.4, 0.6), (0.45, 0.55), (0.5, 0.5), (0.55, 0.45), (0.6, 0.4),(0.65, 0.35), (0.7, 0.3), (0.75, 0.25), (0.8, 0.2), (0.85, 0.15), (0.9,0.1), (0.95, 0.05), and (1.0, 0). When these ratios are independentlychanged, about 100 particles can be distinguished. On the other hand, bythe use of groups of particles which have 15 particle sizes,respectively, of 100 μm to 198 μm that are different by 7 μm each, theparticles in the groups, respectively, are distinguished by their sizes.In the measuring apparatus shown in FIG. 10, the sizes of the smallparticles and fluorescences are measured. In this apparatus, excitinglight is casted in pulses, and long-life phosphorescence emitted by thetag labeling DNA's and fluorescences emitted by the tags labeling theparticles can be measured in distinction from each other in atime-divided manner. Needless to say, the difference in wavelength maybe utilized. In this case, it is preferable to employ fluorescencewavelength ranges different from the phosphorescence wavelength range.Thus, 300 (20×15) kinds in total of DNA probes (particles) aredistinguished at the time of the measurement. As a measuring apparatus,there is used a laser scanning type fluorescence-detecting apparatus ora received-light wavelength selection type cooled CCD. As a wavelengthselector, there can be used a diffraction grating, awavelength-dispersing prism, or a spectroscopic system composed of aplurality of band-pass filters.

[0103] The kinds of the particles are distinguished by the relativeintensities of the fluorescences measured. A large number of particleswith the same particle diameter can be distinguished by varying thewavelength of the exciting light (laser beams). For example, whenparticles are labeled with mixtures of Joe and Tamura (fluorophoresexcitable by means of a YAG laser) in various ratios, about 10 kinds ofparticles can be distinguished by estimating in 10 grades the ratiobetween the intensities of signals obtained from two filters,respectively, adjusted to optimum light-receiving channels for signalsfrom the fluorophores. On the other hand, when particles are labeledwith Rox in addition to Joe and Tamura, 30 kinds in total of particleswith the same particle diameter can be distinguished by the followingmixing ratios of the fluorophores: 10 mixing ratios between Joe andTamura, 10 mixing ratios between Joe and Rox, and 10 mixing ratiosbetween Tamura and Rox. Moreover, when samples are labeled withfive-fluorophores which emit fluorescence having a long wavelength andcan be excited by means of a semiconductor laser, 150 kinds of samplescan be distinguished. Furthermore, when size measurement using particleshaving 15 particle diameters is combined with the above measurements,2,250 kinds of DNA probes can be distinguished. Since the particles holddifferent probes, respectively, on their surfaces, 2,250 kinds of DNA'scan be distinguished and detected. When a container for probes ispartitioned into compartments, or there is used a holding chip having aplurality of sites (compartments) in which the groups, respectively, ofparticles explained above are held, particles holding different groupsof DNA probes can be held in different divisions (compartments), so thatthe number of distinguishable DNA's can easily be increased to 10,000 ormore.

[0104] Although particles are labeled with fluorophore in the aboveexplanation, dyes may be used for the labeling. Since the kinds ofprobes can be freely varied depending on purposes, a multi-probe sensordevice suitable for various purposes can be obtained.

[0105] The above names Joe, Tamura and Rox are trade names given byPerkin-Elmer ABD Corporation, Texas Red is a trade name given byMolecular Probe Co., Ltd., and Cy-5 is a trade name given by AmershamPharmacia Biotech Ltd.

1-31. (Canceled)
 32. A method for manufacturing a probe array foranalyzing target substances comprising: providing a plurality ofreservoirs, each reservoir having particles onto which one kind of probeis immobilized; and supplying each particle into a capillary from eachreservoir in a predetermined order.
 33. A method for manufacturing aprobe array according to claim 32, wherein said each particle issupplied into the capillary by a solution flow.
 34. A method formanufacturing a probe array according to claim 32, wherein eachreservoir contains the particles onto which each different kind of probeis immobilized.
 35. A method for manufacturing a probe array accordingto claim 32, wherein the direction of arrayment of the reservoirs isperpendicular to the capillary.
 36. A method for manufacturing a probearray according to claim 32, wherein said each particle is supplied intothe capillary through a groove for arrayment.